Glutathione is present in almost all biological tissues. Glutathione, a major intracellular reducing substance, plays important roles in catalysis, metabolism, transport and in cellular protection. With respect to cellular protection in particular, glutathione displays its action by (1) capturing and detoxifying harmful free radicals and peroxides produced in the body via the SH group of its own molecule, (2) reducing and deactivating active oxygens such as peroxides by means of glutathione peroxidase, and (3) reacting with toxic compounds by means of glutathione S-transferase and excreting those toxic compounds outside the cell in the form of glutathione conjugates, thereby playing roles in antioxidation, detoxification, protection against radiation injury and increasing temperature resistance, etc.
Thus, when the level of tissue glutathione decreases due to various diseases, ageing and so on, the tissue becomes susceptible to injuries. In such cases, it is important to correct the levels of tissue glutathione to normal values in order to restore cell function. In addition, even in cases when tissue glutathione levels are normal, it is thought that increasing tissue glutathione levels can enhance cell protective functions. In actuality, there are reports which state that glutathione and several thiol compounds were effective when used for the purpose of protecting cells from mutagenic substances and carcinogenic substances as well as reducing the size of animal liver tumors caused by such malignant substances.
Moreover, there has been growing attention in recent years focusing on the correlation between depressed levels of glutathione and the overproduction of active oxygens in relation to ischemia and infarction disorders of various tissues including the heart and brain, as well as various types of lung disorders.
In addition, it is gradually becoming clear that when ischemia is canceled by restoration of blood flow, the formation of active oxygens is accelerated, which brings about even more remarkable decrease in glutathione, thereby resulting in the occurrence of more serious disorders. Similar disorders are observed during cessation or restoration of blood flow during transplantation of various organs such as heart, liver, lungs, pancreas and blood vessels. Those disorders also become problem during incision and removal of organs. Active oxygens and reactive free radicals that are suspected to be the cause of disease are detected both in the cytoplasm of cells that compose tissue and in cellular organelles, especially in mitochondria that produce ATP, which serves as the primary source of energy for cells. It is observed that, in mitochondria, the respiratory chain is the main source of generation of said reactive molecules and the concentration of such has become remarkably elevated during ischemia and reperfusion. In addition, extracellular active oxygens, that are produced on the cell membrane surface of leukocytes that gather at the lesion, are also considered to be harmful to adjacent cells.
The important function of glutathione in the elimination of active oxygens that form during ischemia-reperfusion was first reported in the studies using animals for the disease model (for example, J. Neurochem., Vol. 34, pp. 477-486, 1980; or, Curr. Surgery, January-February edition, pp. 31-33, 1988). Moreover, this was also recognized in human diseases (Circulation, Vol. 81, pp. 201-211, 1990). In each of these cases, the degree of severity of the disease was considered to correlate with the degree of consumption of glutathione.
In addition, it is also reported that in various lung diseases, the severity of the disease state correlates with the decrease in glutathione on the surface of pulmonary epithelial cells (for example, Lung, Vol. 169, pp. 123-138, 1991).
Thus, the significance of replenishing glutathione from outside the body in the prevention and treatment of such diseases would be appreciated, while glutathione itself, the half-life of which in the blood when administered in short, i.e., several minutes, is not very effective in raising tissue glutathione levels. The reason for this is considered that glutathione itself is not efficiently taken up by cells, and glutathione must first be degraded and taken up by cells in the form of peptides and constituent amino acids followed by re-synthesis in the cell.
In an effort to overcome the above-mentioned problems, compounds have been discovered that are superior to glutathion with respect to the ability to elevate tissue glutathione levels. Examples of such compounds include 2-oxothiazolidine-4-carboxylate, .gamma.-L-glutamyl-L-cysteine, and .gamma.-L-glutamyl-L-cysteinyl-glycine ethyl ester (glutathione monoethyl ester). These compounds were discovered through experimentation using human lymphoma cells or animals (for example, Curr. Top. Cell. Regul., Vol. 26, pp. 383-394, 1985; or, Fed. Proc., Vol. 43, pp. 3031-3042, 1984).
The compound represented with general formula (I): ##STR2## wherein R is an ethyl group, is known according to the description of Japanese Examined Patent Publication No. 63-61302; and the compound wherein R is a straight chain, branched or cyclical hydrocarbon group having 3 to 10 carbon atoms, or a straight chain or branched hydrocarbon group having 1 to 5 carbon atoms substituted with an aromatic group is described in Japanese Unexamined Patent Publications No. 64-19059 and No. 2-258727; and the compound wherein R is a methyl group is known according to the description by Flohe, et al. in Z. Klin. Chem. u. Klin. Biochem., Vol. 8, pp. 149-155 (1970), and is also described in Japanese Unexamined Patent Publication No. 64-26516.
Although the documentation of Flohe, et al. indicates that the .gamma.-L-glutamyl-L-cysteine derivative represented with general formula (I) wherein R is a methyl group serves as a substrate of glutathione peroxidase and is used at 20-30% efficiency compared with glutathione, there are no other descriptions whatsoever that suggest other uses of this compound. In contrast, the previously listed Examined Publication or three Unexamined Publications describe that the .gamma.-L-glutamyl-L-cysteine ester derivative indicated in the above-mentioned general formula (I), or its oxidized dimer, is an ester derivative of a glutathione biosynthesis intermediate and has favorable membrane permeability; and that by means of being efficiently incorporated into tissue and subsequently subjected to deesterification followed by promptly biosynthesetic conversion into glutathione, this compound has the effect of elevating the level of tissue glutathione. These publications also describe that this compound is useful as a therapeutic agent for the treatment of liver disease, cataracts and kidney disease.
In addition, with respect to the possibility that, dependent on animals, tissues and dosages used in experiments, excess .gamma.-L-glutamyl-L-cysteine ester derivative incorporated may be present in tissue as unchanged SH compound, the inventors of the present invention also reported that .gamma.-L-glutamyl-L-cysteine ester derivative has various desirable properties: it serves as a good substrate for glutathione peroxidase, glutathione S-transferase and glutathione reductase in its original form, while, unlike glutathione, it is resistant to breakdown by .gamma.-glutamyl transpeptidase (SEIKAQ, Vol. 61, No. 9 (edition containing the abstract of the 62nd meeting of the Japanese Biochemical Society), p. 800, title no. 1a-Ab08, 1989).